Inhibition of agonist-specific desensitization of β2 adrenergic receptors

ABSTRACT

The present invention relates to a method of inhibiting desensitization of a cell to the effects of a compound. The method comprises contacting the cell with an agent capable of inhibiting phosphorylation, by a protein kinase, of a receptor for the compound present on the surface of the cell. The present invention also relates to a method of screening a compound for its ability to inhibit desensitization. The method comprises: i) contacting a receptor specific kinase-containing sample with the compound under conditions such that interaction between receptor specific kinase present in the sample and the compound can occur, and 
     ii) determining the ability of the receptor specific kinase to phosphorylate the receptor for which it is specific.

This is a continuation of application Ser. No. 07/341,983, filed on Apr.24, 1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates, in general, to desensitization, and, inparticular, to a method of inhibiting agonist-specific desensitization,to compounds suitable for use in such a method and to pharmaceuticalcompositions comprising same.

2. Background Information

Desensitization is a general phenomenon which is characterized by areduced responsiveness following prolonged exposure to a hormone ordrug. Occupancy of a wide variety of hormone and neurotransmitterreceptors by agonists often leads to desensitization, that is, to a lossof receptor responsiveness to subsequent stimulation by agonist. Thisparticular phenomenon is generally termed homologous desensitization.This is in contrast to the heterologous form of desensitization, whichis defined as a loss of receptor responsiveness caused by agonistoccupancy of other receptors.

Homologous desensitization has been most thoroughly studied for theβ-adrenergic receptor (βAR)-adenylyl cyclase system (Benovic et al(1988) Ann. Rev. Cell Biol. 4:405-428). Homologous desensitization ofβARs is accompanied by receptor phosphorylation (Sibley et al (1985) J.Biol. Chem. 260:3883-3886; Strasser et al (1986) Biochemistry25:1371-1377). A cAMP-independent kinase, termed βAR kinase, has beendescribed that specifically phosphorylates the agonist-occupied forms ofthe β₂ -adrenergic receptor (β₂ AR) and α₂ -adrenergic receptor (Benovicet al (1986) Proc. Natl. Acad. Sci. USA 83:2797-2801; Benovic et al(1987) J. Biol. Chem. 262:17251-17253) as well as light-activatedrhodopsin (Benovic et al (1986) Nature (London) 322:867-872).Phosphorylation of the β₂ AR by βAR kinase may trigger the process offunctional uncoupling from the stimulatory guanine nucleotide bindingprotein, G_(s) (Benovic et al (1987) Proc. Natl. Acad. Sci. USA84:8879-8882).

While there have been several publications involving attempts atblocking desensitization (Mallorga et al (1980) Proc. Natl. Acad. Sci.USA 77:1341-1345; Shima et al (1983) J. Biol. Chem. 258:2083-2086;Heyworth et al (1984) Biochem. J. 222:189-194; Fano et al (1986) J.Auton. Pharmacol. 6:47-51; DeBernardi et al (1987) Proc. Natl. Acad.Sci. USA 84:2246-2250; and Toews (1987) Mol. Pharmacol. 32:737-742),none of these studies directly demonstrates the inhibition ofagonist-specific desensitization. Moreover, none of the compoundsutilized in these studies directly inhibit the βAR kinase. Theidentification of compounds which specifically inhibit this kinase couldprovide a method of inhibiting desensitization. The inhibition ofdesensitization should result in enhanced and prolonged receptor actionin response to endogenous compounds and drugs. In the case of drugs,this means increased drug efficacy leading to the use of lower drugdoses, and reduced side effects. In addition, it should allow prolongedtreatment with drugs.

SUMMARY OF THE INVENTION

It is a general object of the invention to provide a method of blockingagonist-specific desensitization.

It is a specific object of the invention to provide compounds capable ofinhibiting agonist-specific desensitization.

It is a further object of the invention to provide a pharmaceuticalcomposition comprising as an active ingredient a compound capable ofblocking agonist-specific desensitization.

It is another object of the invention to provide a method of augmentingthe efficacy and duration of treatment of drugs, the continuedadministration of which results in desensitization.

It is a further object of the invention to provide a method of restoringthe effectiveness of receptor-mediated responses to endogenous compoundssuch as hormones and neurotransmitters.

Further objects and advantages of the present invention will becomeclear to one skilled in the art from a reading of the description thatfollows.

In one embodiment, the present invention relates to a method ofinhibiting desensitization of a cell to the effects of a compound. Themethod comprises contacting the cell with an agent capable of inhibitingphosphorylation, by a protein kinase, of a receptor for the compoundpresent on the surface of the cell.

In another embodiment, the present invention relates to a method ofscreening a compound for its ability to inhibit desensitization. Themethod comprises:

i) contacting a receptor specific kinase-containing sample with thecompound under conditions such that interaction between receptorspecific kinase present in the sample and the compound can occur, and

ii) determining the ability of the receptor specific kinase tophosphorylate the receptor for which it is specific.

In a further emobodiment, the present invention relates to an inhibitorof β adrenergic receptor kinase. The inhibitor consists essentially of apeptide corresponding to an intracellular domain of the β₂ adrenergicreceptor.

In yet another embodiment, the present invention relates to apharmaceutical composition. The composition comprises as an activeingredient the above-described peptide (or other agent) in an amountsufficient to exhibit an inhibitory effect on β adrenergic receptorkinase, together with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Inhibition of βAR kinase by polyanions. Urea-treated rod outersegments were phosphorylated by βAR kinase in the presence of theindicated concentrations of polyanion. 100% activity corresponds to 11pmol of phosphate incorporated during a 15-min incubation withoutinhibitor. Δ, dextran sulfate; ∘, heparin; , de-N-sulfated heparin; ▴,polyglutamic acid.

FIGS. 2A-2B (collectively, FIG. 2) Lineweaver-Burk plots for theinhibition of βAR kinase by heparin. FIG. 2A, rhodopsin was varied inthe assay from 3 to 20 μM. The heparin concentrations were 0 (∘), 0.21(), 0.42 (Δ), and 0.63 (▴) μM. FIG. 2B. ATP was varied in the assayfrom 33 to 100 μM. The heparin concentrations were 0 (∘), 0.10 (), 0.21(Δ), and 0.31 (▴) μM.

FIG. 3. Inhibition of βAR kinase by polycations. Urea-treated rod outersegments were phosphorylated by βAR kinase in the presence of theindicated concentrations of polycation. 100% activity corresponds to 13pmol of phosphate incorporated during a 15-min incubation withoutinhibitor. ▴, polylysine; ∘, spermine; , spermidine.

FIG. 4. Reversal of heparin inhibition by polycations. Urea-treated rodouter segments were phosphorylated by βAR kinase in the presence of 1.1μM heparin and the indicated concentration of polycation. 100% activityrepresents phosphorylation in the absence of heparin and corresponds to6 pmol of phosphate incorporated in a 15-min incubation. ▴, polylysine;∘, spermine; , spermidine.

FIGS. 5A-5B (collectively, FIG. 5) Desensitization of β₂ ARs in intact(FIG. 5A) and permeabilized (FIG. 5B) A431 cells. Cells were incubatedfor 10 min with (desensitized) () or without (control) (∘) 1 μM(-)-isoproterenol as detailed. Adenylyl cyclase activities of membranesprepared from the cells were measured in the presence of variousconcentrations of (-)-isoproterenol and were expressed as percent of theactivity in the presence of 10 mM NaF. Values for EC₅₀, maximalstimulation over basal (E_(max), in percent of activity with 10 mM NaF),and activity in the presence of 10 mM NaF (=100%, in pmol of cAMP per mgof protein per min) were as follows: for control intact cells they were200 n (EC₅₀), 56% (E_(max)), and 84±2 pmol/mg/min (100%); fordesensitized intact cells they were 300 nM (EC₅₀), 40% (E_(max)), and70±7 pmol/mg/min (100%) for control permeabilized cells they were 130 nM(EC₅₀), 62% (E_(max)), and 86±2 pmol/mg/min (100%) for desensitizedpermeabilized cells they were 190 nM (EC₅₀), 43% (E_(max)), and 69±3pmol/mg/min (100%). Data are means±SEM of three independent experiments.

FIG. 6. Time course of β₂ AR desensitization in permeabilized A431cells. Permeabilized cells were incubated for the indicated periods oftime with 1 μM (-)-isoproterenol alone (control) or in the presence of100 μM of the peptide β₂ AR-(57-71) [β₂ (57-71)] or 100 nM heparin.Desensitization was measured as the percent loss of stimulation by 10 μM(-)-isoproterenol of adenylyl cyclase activity in membranes. Data aremeans±SEM of six (control) or three independent experiments.

FIGS. 7A-7C (collectively, FIG. 7) Inhibition by heparin of β₂ ARphosphorylation by βAR kinase in a reconstituted system (FIG. 7A) and ofβ₂ AR desensitization in permeabilized A431 cells (FIG. 7B). FIG. 7A:Purified reconstituted β₂ ARs were phosphorylated by purified βAR kinasewith [γ-³² P]ATP in the presence of various concentrations of heparin.Curve-fitting gave an IC₅₀ value of 6.1±2.0 nM. 7C Relevant section of arepresentative autoradiogram. Data are means +SEM of three independentexperiments. FIG. 7B: Permeabilized A431 cells were desensitized byincubation with 1 μM (-)-isoproterenol for 10 min at 37° C. in thepresence of various concentrations of heparin. Desensitization wasmeasured as the percent loss of stimulation by 10 μM (-)-isoproterenolof adenylyl cyclase activity in membranes. Curve-fitting gave an IC₅₀value of 21±4 nM. Data are means±SEM of four independent experiments.

FIGS. 8A-8C (collectively, FIG. 8) Phosphorylation of β₂ ARs inpermeabilized A431 cells. Permeabilized cells were incubated with [γ-³²P]ATP without (CON) or with (ISO) 1 μM (-)-isoproterenol in the absenceor presence of 1 μM heparin. β₂ ARs were solubilized, purified byaffinity chromatography, and electrophoresed on a 10% sodium dodecylsulfate polyacrylamide gel, each lane containing 0.5 pmol of receptor.FIG. 8A: Autoradiogram obtained after a 10-day exposure at -70° C. FIG.8B: Transverse densitometric scan of the autoradiogram. FIG. 8C: Analiquot of purified receptors from cells incubated without(-)-isoproterenol or heparin was photoaffinity-labeled with ¹²⁵I-cyanopindolol diazirine in the absence (CON) or presence (ALP) of 10μM alprenolol, followed by electrophoresis on the same gel (60 fmol perlane) and autoradiography. Similar results were obtained in two otherexperiments.

FIG. 9. Model for the organization of the hamster β₂ AR in the plasmamembrane. The amino acid sequence and proposed structure of the hamsterβ₂ AR in the plasma membrane (Dixon et al (1986) Nature 321:75-79). Thesynthetic peptides used in this study are bracketed and labeled:NT--amino terminus; CI, CII, CIII--first, second and third intracellularloops; EI, EII--first and second extracellular loops; CT-1, CT-2, CT-3--carboxyl terminus.

FIG. 10. Time course of phosphorylation of β₂ AR and synthetic peptidesby βAR kinase. Reconstituted hamster lung β2-adrenergic receptor (0.5pmol) was phosphorylated by βAR kinase (˜30 ng) in the presence () of50 μM (-)-isoproterenol. Two synthetic carboxyl terminal peptides of thehamster lung β₂ AR (CT-2 (Δ), CT-3 (▴)) were also phosphorylated by βARkinase under identical conditions. Following the incubation period thereactions (20 μl) were stopped by addition of 50 μl of SDS sample bufferfollowed by electro-phoresis on a 10% (β₂ AR) or 15% (CT-2, CT-3)homogeneous polyacrylamide gel.

FIG. 11. Kinetic parameters of the βAR kinase for β₂ AR. Reconstitutedβ₂ AR (0.05-0.3 μM) was incubated for 15 min at 30° C. with purified βARkinase (˜30 ng) in the presence (∘) or absence () of 50 μM(-)-isoproterenol. Reactions were stopped by the addition of SDS samplebuffer before electrophoresis on a 10% SDS polyacrylamide gel. ³²P-labeled receptor was determined by cutting and counting the dried gelfollowing autoradiography.

FIG. 12. Kinetic parameters of the βAR kinase for synthetic peptidesCT-2 and CT-3. The synthetic β₂ AR peptides CT-2 (0.6-3.3 mM; Δ) andCT-3 (0.6-4.3 mM; ▴) were incubated for 60 min at 30° C. with purifiedβAR kinase (˜30 ng). Reactions were stopped by the addition of SDS ureasample buffer before electrophoresis on a 9% SDS urea polyacrylamidegel. ³² P-labeled peptide was determined by cutting and counting the gelfollowing autoradiography.

FIG. 13. Inhibition of β₂ AR phosphorylation by synthetic peptides.Reconstituted β₂ AR (˜0.4 pmol) was incubated for 30 min at 30° withpurified βAR kinase (˜30 ng) in the presence of 50 mM Tris-HCl, pH 7.5,2 mM EDTA, 10 mM NaCl, 5 mM MgCl₂, 5 mM sodium phosphate, 50 μM(-)-isoproterenol and 0.10 mM [γ³² P]ATP (2.0 cpm/fmol). Synthetic β₂ ARpeptides were varied from 0 to 1 mM and included CI (Δ), CII (▴), CIII-2(∘) and CT-1 (). Reactions were stopped by the addition of SDSpolyacrylamide gel. ³² P-labeled receptor was determined by cutting andcounting the dried gel following autoradiography.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of inhibiting thedesensitization of a cell to the effects of a compound (i.e. hormone ordrug), the desensitization resulting from the continued contact of thecell with the compound. The method is based on the discovery that theloss of responsiveness of a cell to a particular compound is due tophosphorylation of the cell surface receptor for that compound.

In a specific embodiment, the present invention relates to a method ofblocking the agonist specific (homologous) desensitization of a cell(tissue or organism) that results from prolonged exposure to theagonist. The method comprises contacting the cell (tissue or organism)subject to desensitization with a compound capable of inhibitingphosphorylation of the receptor for a particular agonist.

Compounds suitable for use in the present invention include thosecapable of inhibiting receptor kinases, for example, specific proteinkinases involved in mediating homologous desensitization of adenylylcyclase-coupled receptors (i.e. βAR kinase). (See also Blackshear (1988)FASEB J. 2:2957; Middleton (1988) Ann. Allergy 61:53; Hunnan (1988)Science 243:500.) Examples of such compounds include polyanions,advantageously, acid mucopolysaccharides such as heparin and dextransulfate. Polycations, for example, polylysine, spermine and spermidine,are also capable of inhibiting the receptor specific kinase βAR kinase.

Highly receptor specific inhibition can be achieved using peptidescorresponding to discrete domains of the receptor the phosphorylation ofwhich is sought to be inhibited. For example, the β₂ AR peptides β₂ AR(219-243) (designated CIII-1 below), β₂ AR (56-74) (designated CIbelow), β₂ AR (57-71), β₂ AR (57-71; L→A, N→A), and βAR (57-71; K→A,R→A) are relatively potent inhibitors of βAR kinase, while β₂ AR(97-106) (designated EI below), β₂ AR (137-151) (designated CII below),β₂ AR (248-268) (designated CIII-2 below), β₂ AR (337-355) and β₂ AR(353-381) (designated CT-2 below) and β₂ AR (57-71, E→Q), which peptidesalso inhibit βAR kinase, are somewhat less effective. Methods ofidentifying receptor peptides capable of exerting an inhibitory effecton receptor kinases are set forth in the Examples that follow. Theinvention includes within its scope receptor peptides, for example,those set forth above.

The invention also relates to pharmaceutical compositions comprising asan active ingredient at least one inhibitor of at least one receptorkinase, together with a pharmaceutical carrier. Such compositions caninclude agents (for example, lipophilic reagents) that facilitatetransport of the inhibitor across cell walls. The compositions can be indosage unit for (i.e. pill, tablet or injectable solution, etc.). Theamount of active ingredient to be included in such a composition, andthe amount of active ingredient to be administered, can be determined byone skilled in the art without undue experimentation.

It will be appreciated that the method of the present invention can beused to restore the effectiveness of receptor mediated responses toendogenous compounds, such as hormones and neurotransmitters.

In another embodiment, the present invention relates to a method ofscreening compounds for their ability to block agonist-specificdesensitization. The method can be practiced using a culture of cellspermeabilized according to known protocols (for example, by treatmentwith digitonin), a cell lysate, or a cell free preparation of receptorkinase(s) (all of which will hereinafter be referred to as "receptorkinase containing sample"). The method comprises contacting the receptorkinase containing sample with a compound to be tested for its ability toinhibit desensitization under conditions such that interaction (i.e.binding) of the receptor kinase present in the sample and the compoundcan occur. The ability of the test compound to inhibit receptor kinaseactivity, and, therefore, block agonist-specific desensitization, isthen determined by assaying for the ability of the kinase tophosphorylate the receptor for which it is specific. Assays forphosphorylation activity are known in the art and are exemplified below.

The following non-limiting Examples further describe the presentinvention.

EXAMPLE I Inhibition of βAR Kinase by Polyanions

Heparin (average M_(r) =4000-6000), de-N-sulfated heparin, dextransulfate (average M_(r) =5000), heparin sulfate, chondroitin sulfate B,chondroitin sulfate C, polyaspartic acid (average M_(r) =11,000),polyglutamic acid (average M_(r) =13,600), inositol hexasulfate,inositol hexaphosphate, pyridoxal phosphate, 2,3-diphosphoglycerate,glucosamine 2,6-disulfate, polylysine (average M_(r) =3300), spermine,and spermidine were purchased from Sigma. Frozen bovine retinas werefrom Hormel, while bovine cerebral cortex was obtained from aslaughterhouse.

βAR kinase was purified from bovine cerebral cortex by modification ofprocedure of Benovic et al (J. Biol. Chem. (1987) 262:9026-9032).Briefly, 250 g of bovine cerebral cortex were homogenized with aBrinkmann tissue disrupter and centrifuged (40,000× g, 30 min). Thesupernatant was then precipitated with 13-26% ammonium sulfate. Thismaterial was initially chromatographed on a Ultrogel AcA34 columnequilibrated with 5 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 μg/ml leupeptin,5 μg/ml pepstatin, 10 μg/ml benzamidine, 0.2 mM phenylmethylsulfonylfluoride (buffer A). The peak activity was diluted with an equal volumeof buffer A containing 0.04% Triton X-100 before being applied to aDEAE-Sephacel column. Elution was accomplished with a 0-80 mM NaClgradient in buffer A containing 0.02% Triton X-100. The peak activitywas then applied to a CM-Fractogel column and eluted with a 0-100 mMNaCl gradient in buffer A containing 0.02% Triton X-100. The purifiedkinase is stable at 4° C. for several months. For these studies the βARkinase preparations were >75% pure.

Rod outer segments were prepared from bovine retinas by stepwise sucrosegradient centrifugation (Wilden et al Biochemistry (1982) 21:3014-3022).Rhodopsin kinase-free rod outer segments were prepared by treatment with5 M urea (Shichi et al J. Biol. Chem. (1978) 253:7040-7046) andconsisted of ˜95% rhodopsin, as assessed by Coomassie Blue staining ofsodium dodecyl sulfate-polyacrylamide gels.

Urea-treated rod outer segments were phosphorylated with βAR kinase asfollows. Urea-treated rod outer segments (˜4 μg of rhodopsin) wereincubated with βAR kinase (˜0.1 μg) for 10-15 min at 30° C. in thepresence of 40 mM Tris-HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl₂, 1.5 mM EDTA,and 65 μM ATP (˜1 cpm/fmol) (total reaction volume was 40 μl). Reactionswere stopped by the addition of 40 μl of sodium dodecyl sulfate samplebuffer (see below) followed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. After autoradiography, the phosphorylated rhodopsinbands were excised and counted.

βAR kinase phosphorylates the β₂ AR, the α₂ -adrenergic receptor, andrhodopsin in a stimulus-dependent fashion. Rhodopsin was utilized as thesubstrate in these experiments because of its ease of isolation.However, the inhibitory effects of polyanions observed with βARkinase-catalyzed phosphorylation of rhodopsin have also been observedusing reconstituted β₂ AR as the substrate.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performedby the method of Laemmli (Nature (1970) 227:680-685) using 10%homogeneous polyacrylamide slab gels. Sample buffer contained 8% sodiumdodecyl sulfate, 10% glycerol, 5% 3-mercaptoethanol, 25 mM Tris-HCl, pH6.5, and 0.003% bromphenol blue. After electrophoresis, gels were driedwith a Bio-Rad gel dryer prior to autoradiography.

FIG. 1 demonstrates that a number of polyanions are potent inhibitors ofβAR kinase-catalyzed phosphorylation of rhodopsin. In particular,heparin and dextran sulfate appear to be the most potent. De-N-sulfatedheparin is ˜8-fold weaker suggesting some importance of the N-sulfate.It is interesting to note that the inhibition by heparin, dextransulfate, and de-N-sulfated heparin appears to involve a cooperativeinteraction of the polyanion with βAR kinase, as the data in FIG. 1yield a Hill coefficient of ˜2. Several other related acidmucopolysaccharides, such as heparan sulfate and chondroitin sulfates Band C, are significantly less potent than heparin (Table I). The IC₅₀values of these compounds as well as those of a number of other anioniccompounds are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Effect of polyanions on the activity of βAR kinase                       Phosphorylation of rhodopsin by βAR kinase in the presence of            increasing concentrations of several polyanions was assessed as               described above. The IC.sub.50 is the concentration of compound which         gave 50% inhibition and is presented as the mean ± S.E. with the           number of determinations given in parenthesis. ND, molar                      concentration cannot be accurately determined.                                                IC.sub.50                                                     Compound          μg/ml   μM                                            ______________________________________                                        Heparan           0.77 ± 0.10 (5)                                                                       0.15                                             De-N-sulfated heparin                                                                           6.0 ± 2.4 (3)                                                                         ND                                               Heparan sulfate   1.8        ND                                               Chondroitin sulfate B                                                                           25         ND                                               Chondroitin sulfate C                                                                           6          ND                                               Dextran sulfate   0.76 ± 0.21 (3)                                                                       0.15                                             Polyaspartic acid 14         1.3                                              Polyglutamic acid 27 ± 2 (2)                                                                            2.0                                              Inositol hexasulfate                                                                            12 ± 0.5 (2)                                                                          13.5                                             Inositol hexaphosphate                                                                          3325       3600                                             Pyridoxal phosphate                                                                             222        900                                              2,3-Diphosphoglycerate                                                                          838        1100                                             Glucosamine 2,6-disulfate                                                                       2800       7300                                             ______________________________________                                    

It is interesting that inositol hexasulfate is ˜270 times more potentthan inositol hexaphosphate. This suggests again that the sulfate moietyand not just the anionic character of the molecule is an importantdeterminant in the inhibition of βAR kinase. Several other compoundssuch as pyridoxal phosphate, 2,3-diphosphoglycerate, and glucosamine2,6-disulfate inhibited in the millimolar range. Similar results areobserved when the βAR is used as the substrate with heparin again beingthe most potent inhibitor (IC₅₀ ˜0.03 μM, data not shown).

Since heparin is the most potent inhibitor, the kinetics of heparininhibition of βAR kinase were further characterized.

FIG. 2A shows a Lineweaver-Burk plot of data where the rhodopsinconcentration is varied in the presence of several differentconcentrations of heparin. In the absence of heparin, a K_(m) =5.3 μMwas obtained. Heparin appears to be a competitive inhibitor with respectto rhodopsin. A replot of these data (slope versus heparinconcentration, not shown) yields a K_(i) ˜0.5 μg/ml (˜0.10 μM). FIG. 2Bshows a similar plot when the ATP concentration is varied in thepresence of several different concentrations of heparin. These datasuggest that heparin is a mixed type inhibitor with respect to ATP.

Polyanions are known to inhibit casein kinase II, whereas polycations,such as spermine, spermidine, and putrescine, are able to activatecasein kinase II at low Mg²⁺ or at 50-100 mM KCl concentrations(Hathaway et al (1982) Curr. Top. Cell. Regul. 21:101-127). In contrast,spermine and spermidine are weak inhibitors of βAR kinase (FIG. 3).Polylysine is more potent with an IC₅₀ ˜69 μM. Similar results areobserved at low Mg² + concentrations, as well as when 50 mM KCl isincluded in the incubation.

At lower concentrations, polycations are able to partially reverse theinhibition of βAR kinase by heparin (FIG. 4). Polylysine is mosteffective with an EC₅₀ ˜2.8 μM and a recovery of ˜100% of the initialuninhibited activity. Spermine and spermidine also reverse theinhibition with EC₅₀ values of 40 and 630 μM and recoveries of ˜68 and57% activity, respectively. At higher concentrations these polycationsare again inhibitory to the system. The mechanism of this reversal mostlikely represents a direct interaction of the polycation with heparin.

In summary, these results indicate that polyanions, in particularheparin and dextran sulfate, are potent inhibitors of the β-adrenergicreceptor kinase. Polycations such as polylysine, spermine, andspermidine also inhibit βAR kinase activity. Polycations are also ableto reverse the inhibitory effects of heparin.

EXAMPLE II Inhibition of βAR Kinase Prevents Rapid HomologousDesensitization of β₂ -ARs

[α-³² P]ATP, [γ-³² P]ATP, [³ H]cAMP, 125I-labeled cyanopindolol (¹²⁵I-cyanopindolol), and ¹²⁵ I-cyanopindolol diazirine were obtained fromNew England Nuclear; heparin (H-3125 from porcine mucosa), from Sigma.1-5(isoquinoline sulfonyl)-2-methylpiperazine (designated H-7), fromCalbiochem; and digitonin, from Gallard Schlessinger. The peptidecorresponding to residues 1-24 of the heat-stable inhibitor ofcAMP-dependent protein kinase [PKI-(1-24) tetracosapeptide: Scott et alProc. Natl. Acad. Sci. USA (1986) 83:1613-1616] and the β₂ AR-(57-71)pentadecapeptide(Ala-Ile-Ala-Lys-Phe-Glu-Arg-Leu-Gln-Thr-Val-Thr-Asn-Tyr-Phe; Kobilka etal Proc. Natl. Acad. Sci USA (1987) 84:46-50) and β₂ AR-(59-69)undecapeptide (Ala-Lys-Phe-Glu-Arg-Leu-Gln-Thr-Val-Thr-Asn) werechemically synthesized.

Purified β₂ AR was phosphorylated by purified βAR kinase as follows. β₂ARs from hamster lung were purified by affinity chromatography and HPLCto >95% homogeneity (Benovic et al (1984) Biochemistry 23:4510-4518).βAR kinase was purified from bovine cerebral cortex by precipitationwith ammonium sulfate, followed by chromatography on Ultrogel AcA34,DEAE-Sephacel, and CM-Fractogel to >75% purity as described (see ExampleI and Benovic et al (1987) J. Biol. Chem. 262:9026-9032). Purified β₂ARs were inserted into phosphatidylcholine vesicles by chromatography onExtracti-gel, followed by polyethylene glycol treatment andcentrifugation at 280,000× g for 90 min (Cerione et al (1984)Biochemistry 23:4519-4525). Phosphorylation of the reconstitutedreceptors by purified βAR kinase was done in the presence of 50 μM(-)-isoproterenol as described (Benovic et al (1987) J. Biol. Chem.262:9026-9032). Subsequently, the samples were subjected to sodiumdodecyl sulfate polyacrylamide gel electrophoresis on 10% gels (Laemmli(1970) Nature 227:680-685). The phosphorylated β₂ ARs were visualized byautoradiography, and the corresponding bands were cut out and theircontent of ³² P quantified.

Permeabilization of cells was accomplished as follows. Human epidermoidcarcinoma A431 cells were grown to about 95% confluence in Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serum. Cellswere harvested with collagenase, washed three times with calcium-freephosphate-buffered saline (PBS), then washed twice in 150 mM potassiumglutamate/10 mM Hepes/5 mM EGTA/7 mM MgCl₂, pH 7.1 (KG buffer), andfinally resuspended in KG buffer supplemented with 5 mM glucose and 2 mMATP (KG-A buffer) at a density of 4×10⁷ cells per ml.

The concentration of digitonin required to permeabilize the cells wasfound to vary considerably with cell density. At 4×10⁷ cells per ml,about 0.015% digitonin was necessary to achieve permeabilization of >95%cells as assessed by staining with trypan blue. In all experiments,digitonin was added stepwise until >95% of the cells were trypan bluepositive.

Desensitization of β₂ ARs in A431 cells was effected as follows.Permeabilized cells in KG-A buffer (or, for controls, intact cells inPBS) were incubated with or without 1 μM (-)-isoproterenol for 10 min orthe indicated times (4×10⁷ cells per ml). The incubation was terminatedby addition of 10 vol of ice-cold KG buffer (or PBS, respectively),followed by centrifugation at 1000× g for 5 min. After three identicalwashes, cells were disrupted with a Polytron homogenizer in 5 mMTris-HCl, pH 7.4/2 mM EDTA. Crude membranes were prepared by spinningthe supernatant after low-speed centrifugation (1000× g for 5 min) at40,000× g for 20 min.

Adenylyl cyclase activity in the membranes was determined as describedby Salomon et al (Anal. Biochem. (1974) 58:541-548). The free Mg²⁺concentration in the assay was 4 mM. The incubation lasted for 20 min at37° C.; accumulation of [³² P]cAMP was linear over this time period.

β₂ ARs in permeabilized A431 cells were phosphorylated as follows.Permeabilized cells (4×10⁸) in 10 ml of KG-A buffer containing 1 mCi (1Ci=37 GBq) of [γ-³² P]ATP were incubated with or without 1 μM(-)-isoproterenol (and other compounds as indicated) for 10 min at 37°C. All buffers used in these experiments contained 5 μg of soybeantryspin inhibitor, 5 μg of leupeptin, and 10 μg of benzamidine per mland 0.1 mM phenylmethylsulfonyl fluoride. The reaction was terminated byaddition of 30 ml of 150 mM potassium glutamate/5 mM EDTA/10 mM sodiumphosphate, pH 7.1, at 0° C. and centrifugation at 1000× g for 5 min.After three identical washing steps, crude membranes were prepared asabove. The membranes were solubilized with 2% digitonin, and 62 ₂ ARswere purified by affinity chromatography with alprenolol-Sepharose(Benovic et al (1984) Biochemistry 23:4510-4518). Equal amounts of β₂ARs from each sample (≈0.5 pmol, determined by radioligand binding using¹²⁵ I-cyanopindolol) were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described above. Gels were fixedin 40% (vol/vol) methanol and 15% (vol/vol) acetic acid and dried priorto autoradiography.

In addition, purified β₂ ARs in these experiments were identified byphotoaffinity labeling with 125I-cyanopindolol diazirine. Aliquots ofthe samples (≈60 fmol of β₂ ARs) were desalted over Sephadex G-50 andincubated for 2 hr at 20° C. with 200 pM of the ligand with or without10 μM alprenolol. Free ligand was removed by desalting as above, and thesamples were irradiated with UV light for 3 min. The samples were loadedon the same gel as the samples described above.

Concentration-response curves were analyzed by nonlinear curve-fittingto the Hill equation as described (Lohse et al (1986) Mol. Pharmacol.30:403-409). Adenylyl cyclase activity was expressed as the percentageof the activity in the presence of 10 mM NaF. Since NaF stimulatesadenylyl cyclase via the stimulatory guanine nucleotide binding protein,G_(s), this normalizes for effects that occur at the level of G_(s) orthe cyclase (i.e., which effects represent heterologousdesensitization). Desensitization was assessed by measuring the loss ofmaximal stimulation by isoproterenol [determined with 10 μM(-)-isoproterenol] and was calculated as [1-stimulation(desensitized)/stimulation (control)]×100. For example, a decrease from60% to 40% of NaF-stimulated activity corresponds to a desensitizationof [1-(40/60)]×100=33%.

A variety of permeabilization techniques were tested for their abilityto provide constant and reproducible access to the cytosol while leavinghomologous desensitization unaltered. These included scrape-loading(McNeil et al (1984) J. Cell Biol. 98:1556-1564),electro-permeabilization (Knight et al (1986) Biochem. J. 234:497-506),and permeabilization with staphylococcal α toxin (Fussle et al (1981) J.Cell Biol. 91:83-94) and different detergents. Permeabilization withdigitonin gave the most consistent results and was used for all futureexperiments.

FIG. 5 shows that permeabilization of A431 cells with digitonin did notchange the pattern of homologous desensitization to isoproterenol (1μM). Isoproterenol-induced stimulation of adenylyl cyclase in membranesfrom cells incubated with 1 μM isoproterenol for 10 min at 37° C. wasreduced in both potency and maximal effect as compared with membranes ofcontrol cells. In both intact and permeabilized cells, the extent ofmaximal stimulation over basal activity was reduced by the pretreatmentfrom ≈60% to ≈40% of NaF-stimulated activity, corresponding to adesensitization of ≈30%.

FIG. 6 shows the time course of desensitization in permeabilized cellsin the absence or presence of two inhibitors of βAR kinase, heparin (100nM) and a peptide corresponding to the first intracellular loop of thehuman β₂ AR [β₂ AR-(57-71), 100 μM]. In the absence of inhibitors,desensitization occurred with a half-time of <5 min. β₂ AR-(57-71)markedly slowed the rate of desensitization, and desensitization wasalmost completely abolished by 100 nM heparin. Neither compound affecteddesensitization in intact cells (data now shown).

It has been shown that heparin and its analogues may alter stimulationof adenylyl cyclase at micromolar concentrations (Amsterdam et al (1978)Biochim. Biophys. Acta 544:273-283). To ascertain that the inhibition ofdesensitization by heparin was not due to a reduced formation of cAMP,the accumulation of [³² P]cAMP from [α-³² P]ATP added to permeabilizedcells was determined. The presence of up to 1 μM heparin did not reduce[³² P] CAMP formation either in the presence or the absence ofisoproterenol. Thus, the data indicate that compounds that inhibit βARkinase slow down or suppress homologous desensitization.

To show that these effects are indeed due to inhibition of βAR kinase,the concentration dependence for inhibition of βAR kinase activity in areconstituted system was compared with that for inhibition of β₂ ARdesensitization by heparin (FIG. 7). Heparin inhibited phosphorylationof pure reconstituted β₂ ARs by βAR kinase with an IC₅₀ value of 6 nMand caused almost complete inhibition at 30-100 nM (FIG. 7A).Desensitization of β₂ ARs in permeabilized A431 cells was inhibited byheparin with an IC₅₀ value of 20 nM, and inhibition was virtuallycomplete at concentrations above 100 nM. Thus, theconcentration-response curves for the two effects are similar.

If the inhibition of desensitization by heparin is in fact caused byinhibition of βAR kinase, then it would be expected that heparin wouldreduce the isoproterenol-induced phosphorylation of β₂ ARs inpermeabilized cells. Therefore, permeabilized A431 cells were incubatedwith [γ-³² P]ATP with or without 1 μM isoproterenol in the absence orpresence of 1 μM heparin. Incubation time (10 min) and conditions werethe same as in the desensitization experiments reported in FIG. 7. Inthe absence of heparin, isoproterenol caused a 5-fold increase in thephosphorylation of the β₂ ARs (FIG. 8). Heparin (1 μM) markedly reducedthis increase while not affecting basal phosphorylation. Quantitation byscanning of the autoradiograms showed that heparin reduced theisoproterenol-induced phosphorylation by 50-80% in three experiments(FIG. 8B). Although some receptor degradation appeared to occur (asevidenced by a band of lower molecular weight), photoaffinity labelingwith ¹²⁵ I-cyanopindolol diazirine confirmed that the purifiedphosphorylated bands represent β₂ ARs (FIG. 8C). Thus, inhibition of β₂AR desensitization by heparin is paralleled by an inhibition ofisoproterenol-induced receptor phosphorylation.

Finally, the effects of other kinase inhibitors and of analogues of theβ₂ AR peptide both on phosphorylation of reconstituted β₂ ARs by βARkinase and on desensitization in permeabilized A431 cells were compared(Table II). The peptide PKI-(1-24), which inhibits cAMP-dependentprotein kinase with a K_(i) value of 5×10⁻⁹ M (Scott et al (1986) Proc.Natl. Acad. Sci. USA 83:1613-1615), had no effect at concentrations upto 1 μM on either βAR kinase activity or desensitization. H7, aninhibitor of protein kinase C and cyclic nucleotide-dependent proteinkinases with K_(i) values of about 5 μM (Hidaka et al (1984)Biochemistry 23:5036-5041), also did not affect either βAR kinaseactivity or desensitization at 1 μM and 10 μM. At 100 μM it caused ≈20%inhibition of both processes.

                  TABLE II                                                        ______________________________________                                        Inhibition of βAR kinase and of desensitization                          by kinase inhibitors and peptides of the β.sub.2 AR                                            Inhibition Inhibition                                                         of         of                                                      Concentration                                                                            βAR kinase,                                                                         Desensitization,                             Compound   μM      %          %                                            ______________________________________                                        Kinase inhibitors                                                             Heparin    0.1        96 ± 1  102 ± 16                                  PKI-(1-24) 1          0 ± 9    0 ± 12                                   H7         1          3 ± 4   0 ± 4                                                10         2 ± 7   7 ± 5                                                100        19 ± 6  22 ± 14                                   β.sub.2 AR peptides                                                      β.sub.2 AR-(57-71)                                                                  100        80 ± 13 52 ± 12                                   β.sub.2 AR-(59-69)                                                                  100        0 ± 6    3 ± 12                                   ______________________________________                                         Inhibition of βAR kinase and desensitization were measured as shown      in FIG. 7. βAR kinase activity under control conditions corresponds      to 6.5 ± 1.6 pmol of phosphate incorporated during a 30min incubation.     Desensitization under control conditions was 29 ± 4%. Data are means       ± SEM of at least three experiments.                                  

While the 15-amino acid peptide representing the first intracellularloop of the human β₂ AR at a concentration of 100 μM caused significantinhibition of βAR kinase activity, the central 11-amino acid segment wasvirtually inactive in this respect. In parallel, the 15-amino acidpeptide markedly inhibited desensitization (see also FIG. 6), whereasthe shorter peptide did not affect it. These data confirm a correlationbetween inhibition of βAR kinase and inhibition of homologousdesensitization.

EXAMPLE III Synthetic Peptides of the Hamster β₂ AR as Substrates andInhibitors of the βAR Kinase

Peptides were synthesized by tBOC chemistry on an Applied Biosystems430A Synthesizer. Peptides were deblocked by HF treatment and werepurified by reverse phase high performance liquid chromatography on aC18 column using a 0-50% acetonitrile gradient.

The β₂ -adrenergic receptor from hamster lung was purified to apparenthomogeneity by sequential affinity and high performance liquidchromatography as described (Benovic et al (1984) Biochemistry23:4510-4518). The purified receptor was reinserted intophosphatidylcholine vesicles as previously described (Cerione et al(1983) Nature 306:562-566). The protein-lipid pellets were resuspendedin 20 mM Tris-HCl, pH 7.2, 2 mM EDTA and used as a substrate for the βARkinase.

βAR kinase was purified from bovine cerebral cortex by modification of apreviously described procedure (Benovic et al (1987) J. Biol. Chem.262:9026-9032). Briefly, 250 g of bovine cortex was homogenized and theresulting high speed supernatant fraction was precipitated with 13-26%ammonium sulfate. This material was initially chromatographed on anUltrogel AcA34 column equilibrated with 5 mM Tris-HCl, pH 7.5, 2 mMEDTA, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 15 μg/ml benzamidine, 0.2 mMphenylmethyl sulfonyl fluoride (buffer A). The peak activity was diluted1:1 with buffer A containing 0.02% triton X-100 and applied to a DEAESephacel column. Elution was accomplished with a 0-80 mM NaCl gradientin buffer A containing triton X-100. The peak activity was then applieddirectly to a CM Fractogel column and eluted with a 0-100 mM NaClgradient in buffer A containing triton X-100. The purified kinase wasstored at 4° C.

Phosphorylation of βAR was accomplished by incubating reconstituted βAR(0.5-5 pmol/incubation) with βAR kinase (10-50 ng) in a buffercontaining 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM NaCl, 5 mM MgCl2, 5mM sodium phosphate, 0.5 mM ascorbic acid, 0.15 mM [γ-³² P]ATP (1-5cpm/fmol) at 30° C. for the indicated time (see Figures indicated belowand corresponding description). Some incubations also contained 50 μM(-)-isoproterenol. Incubations were stopped by the addition of 50 Al ofSDS sample buffer followed by electrophoresis on 10% homogeneouspolyacrylamide gels. Phosphorylated βAR was visualized byautoradiography and the corresponding bands were excised and counted todetermine the pmol of phosphate incorporated.

Synthetic βAR peptides were phosphorylated as follows by βAR kinase.Synthetic peptides derived from the hamster lung β₂ AR were incubatedwith βAR kinase (50-100 ng) in a buffer containing 50 mM Tris-HCl, pH7.5, 2 mM EDTA, 10 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.10 mM[(γ-32P]ATP (1-5 cpm/fmol) at 30° C. for the indicated time (see Figuresindicated below and corresponding descriptions). The peptideconcentrations varied from 0.5-6 mM. Peptides were separated from freeATP by one of several methods. In most experiments the reactions werestopped by the addition of 50 μl of SDS sample buffer or SDS urea samplebuffer followed by electrophoresis on either a 15% homogeneouspolyacrylamide gel or a 9% polyacrylamide gel containing 6.5 urea (seebelow). Following autoradiography the phosphorylated peptides wereexcised and counted to determine the amount of phosphate incorporated.In some experiments, samples were separated by reverse phase highperformance liquid chromatography on a C18 column (Kuenzel et al (1985)Proc. Natl. Acad. Sci. USA 82:737-741). Following injection of thesample the column was washed with 30 ml of buffer (0.1 M sodiumphosphate, pH 6.5, 0.1 M NaCl) before elution with a linear gradientfrom 0 to 50% acetonitrile. An alternative method involved directapplication of the sample to phosphocellulose paper followed byextensive washing in 75 mM H₃ PO₄ (Cook et al (1982) Biochemistry21:5794). While this procedure gave comparable results for the CT-2peptide, the CT-3 peptide did not appear to bind appreciably to thephosphocellulose paper.

The inhibition of substrate phosphorylation by βAR kinase in thepresence of synthetic peptides was assayed as follows. Reconstituted βAR(0.2-1 pmol/incubation) was incubated with βAR kinase (10-30 ng) in abuffer containing 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM NaCl, 5 mMMgCl₂, 5 mM sodium phosphate, 50 μM (-)-isoproterenol, 0.10 mM [γ-³²P]ATP (1-3 cpm/fmol) at 30° C. for 30 min. Synthetic β₂ AR peptides werevaried in concentration from 0 to 2 mM. Reactions were quenched by theaddition of SDS sample buffer followed by gel electrophoresis. In someexperiments urea treated rod outer segments (Wilden et al (1982)Biochemistry 21:3014-3022; Shichi et al (1978) J. Biol. Chem.253:7040-7046) were used as the substrate for βAR kinase. To furtherdefine the specificity of the peptide inhibition, phosvitin, casein (seeExample I) and the synthetic peptides CT-2 and CT-3 were used assubstrates for βAR kinase.

SDS polyacrylamide gel electrophoresis was performed by the method ofLaemmli (Nature (1970) 227:660-685) using 10 or 15% homogeneous slabgels. SDS sample buffer contained 8% SDS, 10% glycerol, 5%β-mercaptoethanol, 25 mM Tris-HCl, pH 6.5 and 0.003% bromphenol blue.SDS urea polyacrylamide gel electrophoresis was carried out using 6.5 Murea, 0.1% SDS, 100 mM H₃ PO₄, pH 6.8 with Tris-HCl and 9% acrylamide(Swank et al (1971) Anal. Biochem. 39:462). SDS urea sample buffercontained 1% SDS, 8M urea, 10 mM H₃ PO₄, pH 6.8 with Tris-HCl, 1%β-mercaptoethanol and 0.003% bromphenol blue.

FIG. 9 presents the amino acid sequence and proposed topology of thehamster lung β₂ AR. The synthetic peptides utilized in this study arehighlighted and encompass the amino and carboxyl termini as well as thefirst and second extracellular and first, second and third intracellularloops.

Initial studies focused on determining whether any of these 10 peptidescould serve as substrates for βAR kinase. The results shown in FIG. 10demonstrate that two of the carboxyl terminal peptides (CT-2 and CT-3)are phosphorylated by βAR kinase. These peptides are, however, muchpoorer substrates than the agonist-occupied β₂ AR even when present at a100,000-fold higher concentration. Phosphoamino acid analysis of thephosphorylated peptides reveals that CT-2 contains solely phosphoserinewhile CT-3 contains predominately phosphoserine with somephosphothreonine (data not shown). None of the other peptides tested arephosphorylated by βAR kinase, however, two of these peptides (CIII-2 andCT-1), which contain a consensus sequence for cAMP dependent proteinkinase phosphorylation, serve as substrates for protein kinase A.

The kinetics of β₂ AR phosphorylation by βAR kinase in the presence orabsence of the β-agonist isoproterenol are shown in FIG. 11. It isevident that the major agonist-promoted difference in thephosphorylation of the receptor is in the Vmax, with theagonist-occupied receptor having an -6 fold higher Vmax (37 vs 6.6 nmolPi/min/mg, Table III). In contrast, the Km of the receptor varies only1.8-fold (0.16 vs 0.29 μM). Overall, the agonist-occupied receptor is an˜10-fold better substrate than the unoccupied receptor as assessed bythe Vmax/Km ratio. In contrast, the phosphorylation of the syntheticBEAR peptides by βAR kinase have strikingly different kinetics (FIG.12). The carboxyl peptide CT-2 is phosphorylated by βAR kinase with a Km˜5 mM and a Vmax ˜2.8 nmol Pi/min/mg while CT-3 has a Km ˜8 mM and aVmax ˜2.4. A comparison of the Vmax/Km ratios demonstrates that theagonist-occupied receptor is an ˜10⁶ -fold better substrate than thecarboxyl peptides (Table III). The major difference between thesubstrates (receptor vs. peptides) is in the Km obtained with a32,000-50,000 fold difference.

                  TABLE III                                                       ______________________________________                                        βAR kinase phosphorylation of β.sub.2 AR and synthetic              β.sub.2 AR peptides                                                                Km     Vmax                                                         SUBSTRATE (μM)                                                                              (nmol/min/mg)                                                                             Vmax/Km                                                                              Ratio                                     ______________________________________                                        β.sub.2 AR + ISO                                                                   0.16   37          231    1                                         β.sub.2 AR                                                                         0.29   6.6          23    10.sup.-1                                 CT-2      5200   2.8         5.4 × 10.sup.-4                                                                2.3 × 10.sup.-6                     CT-3      7900   2.4         3.0 × 10.sup.-4                                                                1.3 × 10.sup.-6                     ______________________________________                                    

Overall, these results demonstrate that the intact β₂ AR serves as amuch better substrate for βAR kinase than the peptides suggesting thatthe secondary or tertiary structure of the receptor is important forkinase recognition. These results also indicate that βAR kinaserecognizes other regions of the receptor in addition to thephosphorylation sites at the carboxyl terminus.

To address whether other regions of the β₂ AR might interact with βARkinase, the synthetic peptides were tested as potential inhibitors of β₂AR phosphorylation. As shown in FIG. 13 a number of synthetic peptidesare potent inhibitors of βAR phosphorylation by βAR kinase. Theseinclude the first intracellular loop (C-I) which has an IC₅₀ ˜40 μM aswell as the second and third intracellular loops (CII, CIII-1 andCIII-2) and the carboxyl terminus (CT-1 and CT-2) which have IC₅₀ sranging from 70-320 μM (Table IV). Several peptides do not inhibit thephosphorylation when tested at a concentration of ˜0.5 mM (NT, E-II, andCT-3). These results indicate that βAR kinase may interact with multipleregions of the receptor, perhaps explaining the ˜30,000-fold differencein Km between the intact receptor and synthetic peptides forphosphorylation by βAR kinase (Table III).

                  TABLE IV                                                        ______________________________________                                        Synthetic peptides of the hamster β2AR as                                inhibitors of β.sub.2 AR phosphorylation by βAR kinase                               IC.sub.50                                                           Peptide   (μM)                                                      ______________________________________                                               NT (17-32)                                                                              ND                                                                  CI (56-74)                                                                               40                                                                 EI (97-106)                                                                             300                                                                 CII (137-151)                                                                           240                                                                 EII (177-190)                                                                           ND                                                                  CIII-1 (219-243)                                                                         76                                                                 CIII-2 (248-268)                                                                        208                                                                 CT-1 (337-355)                                                                          320                                                                 CT-2 (353-381)                                                                          142                                                                 CT-3 (396-418)                                                                          ND                                                           ______________________________________                                         ND  no significant inhibition when tested at a concentration of ˜50     μM.                                                                   

Since the first intracellular loop β₂ AR peptide was the most potentinhibitor of β₂ AR phosphorylation by βAR kinase the specificity of thisinhibition was studied further. As the length of the peptide decreasesthe ability to inhibit βAR kinase also decreases (Table V). Inparticular, shortening of the peptide from 15 to 11 amino acids resultsin a dramatic loss of the inhibition (IC₅₀ increases from 62 to 2600μM). This indicates that one or more of the four amino acids removed(A,I,Y,F) is critical for the inhibition. Several mutant peptides withamino acid substitutions have also been synthesized. A peptide withLeu⁶⁴ and Asn⁶⁹ both changed to alanine does not appear to affect theinhibition. Converting Lys⁶⁰ and Arg⁶³ to alanine also does notsignificantly affect the inhibition. In contrast, a peptide which hasGlu⁶² changed to glutamine has significantly lower affinity as aninhibitor (IC₅₀ from 62 to 280 μM) suggesting that the glutamic acid isimportant in kinase interaction. Also shown in Table V are results fromstudies with peptides derived from the first intracellular loops of theβ1-adrenergic receptor, the α2-adrenergic receptor and rhodopsin. It isevident that these peptides do not significantly inhibit β₂ ARphosphorylation by βAR kinase. Interestingly the β₁ AR peptide differsby only four amino acids (out of 15) from the β₂ AR peptide. However,one of these amino acids is Glu⁶² which appears to be important forinhibition.

                  TABLE V                                                         ______________________________________                                        Comparison of first intracellular loop peptides                               as inhibitors of βAR kinase                                                                              IC.sub.50                                     Peptide             Sequence          (μM)                                 ______________________________________                                        β.sub.2 AR (56-74)                                                                      TAIAKFERLQTVTNYFITS                                                                            40                                            β.sub.2 AR (57-71)                                                                                    AIAKFERLQTVTNYF                                                                      62                                        β.sub.2 AR (59-69)                                                                                    AKFERLQTVTN                                                                             1600                                   β.sub.2 AR (60-66)                                                                                    KFERLQT                                                                                     2600                               β.sub.2 AR (57-71, L→A, N→A)                                              AIAKFERAQTVTAYF   43                                           β.sub.2 AR (57-71, K→A, R→A)                                               AIAAFEALQTVTNYF      88                                       β.sub.2 AR (57-71, E→Q)                                                                   AIAKFQRLQTVTNYF                                                                           280                                       β.sub.1 AR                                                                                                   700AKTPRLQTLTNLF                          α.sub.2 AR                                                                                                >1000FTSRALKAPQNLF                          Rhodopsin                         >1000   VTVQHKKLRTPLNYI                     ______________________________________                                    

Further confirming the specificity of the peptide inhibition is theobservation that the peptides failed to inhibit βAR kinase mediatedphosphorylation of non-receptor substrates such as casein and phosvitin.Moreover, the first, second and third intracellular loop peptides didnot inhibit βAR kinase phosphorylation of the carboxyl terminal βARpeptides.

For purposes of completing this disclosure, the entire contents of allpublications cited hereinabove, are hereby incorporated by reference.

It will be clear to one skilled in the art to which the presentinvention relates from a reading of the above disclosure that certainchanges in form and detail can be made without departing from the truescope of the invention.

What is claimed is:
 1. A method of inhibiting desensitization of a cellto the effects of a compound, comprising causing the cell to becontacted with a pharmaceutically acceptable agent that inhibits β2adrenergic receptor protein kinase and thereby inhibits β2 adrenergicreceptor protein kinase-induced phosphorylation of β2 adrenergicreceptors for said compound, which β2 adrenergic receptors are presenton the surface of said cell, wherein said contacting is effected underconditions such that said inhibition of desensitization is effected. 2.The method according to claim 1 wherein said desensitization ishomologous desensitization.
 3. The method according to claim 1 whereinsaid compound is endogenous to said cell.
 4. The method according toclaim 1 wherein said compound is a drug.
 5. The method according toclaim 1 wherein said cell is a human cell.
 6. The method according toclaim 1 wherein said agent is a polyanion.
 7. The method according toclaim 1 wherein said agent is a peptide having the amino acid sequenceof a fragment of a β2 adrenergic receptor present on the cell or apeptide having the β2 adrenergic receptor kinase inhibitory functionthereof.
 8. A method of inhibiting desensitization of a cell to theeffects of a compound, comprising causing the cell to be contacted witha pharmaceutically acceptable agent that inhibits β2 adrenergic receptorprotein kinase and thereby inhibits β2 adrenergic receptor proteinkinase-induced phosphorylation of β2 adrenergic receptors for saidcompound, which β2 adrenergic receptors are present on the surface ofsaid cell, wherein said contacting is effected under conditions suchthat said inhibition of desensitization is effected; and wherein saidagent is a polyanion or a peptide derived from a β2 adrenergic receptor.